Assessment of environmental safety and protective efficacy of O-antigen deficient DIVA capable Salmonella Enteritidis against chicken salmonellosis.

In this study, we incorporated deletion of the O-antigen ligase gene to an attenuated Salmonella Enteritidis (SE) strain, JOL919 (SE PS; Δlon ΔcpxR), using the Lambda-Red recombination method and evaluated the safety and immunological aspects of the novel genotype, JOL2381 (SE VS: Δlon, ΔcpxR, ΔrfaL). Assessment of fecal shedding and organ persistence following administration via oral and IM routes revealed that the SE VS was safer than its parent strain, SE PS. Immunological assays confirmed that immunization via the oral route with SE PS was superior to the SE VS. However, chickens immunized with SE PS and SE VS strains via the IM route showed higher humoral and cell-mediated immune responses. Compared to PBS control, the IM route of immunization with SE VS resulted in a higher IgY antibody titer and expansion of CD4+ and CD8+ T-cell populations, which resulted in the clearance of Salmonella from the liver and splenic tissues. Furthermore, deletion of the O-antigen ligase gene caused lower production of LPS-specific antibodies in the host, promoting DIVA functionality and making it a plausible candidate for field utilization. Due to significant protection, high attenuation, and environmental safety concerns, the present SE VS strain is an ideal choice to prevent chicken salmonellosis and ensure public health.

Multistate outbreak of turtle-associated salmonellosis highlights ongoing challenges with the illegal sale and distribution of small turtles.

The sale and distribution of small turtles (shell length <4 inches) as pets has been banned in the United States since 1975 because of the risk of Salmonella transmission, especially to children. Despite this 48-year-old ban, salmonellosis outbreaks continue to be linked to contact with small turtles. During investigations of turtle-associated outbreaks, information regarding the turtle farm of origin is difficult to obtain because turtles are commonly sold by transient vendors. During 2020-2021, public health officials investigated a multistate illness outbreak caused by Salmonella enterica serotype Typhimurium linked to pet small turtles. Cases were defined as a laboratory-confirmed Salmonella Typhimurium infection highly related (within 0-6 allele differences) to the outbreak strain based on whole-genome sequencing analysis by core-genome multilocus sequence typing with illness onset occurring during 27 August 2020-14 May 2021. Forty-three patients were identified from 12 states; of these, 35% (15/43) were children <5 years old. Among patients with available information, 37% (14/38) were hospitalized, and one death was reported. Seventy-four percent (25/34) of patients reported turtle exposure in the week before illness onset, and 84% (16/19) specified exposure to small turtles. The outbreak strain was isolated from samples collected from a Pennsylvania patient's small turtle tank. Two patients reported purchasing their small turtles from pet stores. Salmonella Braenderup was isolated from samples collected from small turtles and their habitat at one of these stores; however, at that time, this strain was not associated with any human illnesses. This investigation was notable because of the documented sale of small turtles from several pet stores combined with the identification of a single small turtle supplier to these pet stores. The high proportion of children involved in this outbreak highlights the continued need to educate the pet industry as well as parents and caregivers about the risk of turtle-associated salmonellosis especially in children. Understanding and addressing the persisting challenges related to the illegal sale and distribution of small turtles could reduce the burden of turtle-associated salmonellosis.

Salmonella assessment along the Spanish food chain: Likelihood of Salmonella occurrence in poultry and pig products is maintained across the food chain stages.

Salmonellosis is one of the most important foodborne diseases worldwide, including the European Union. Despite the One Health approach measures for risk assessment and risk management implemented by the European Union, the occurrence of disease and disease outbreaks remains high (e.g. 694 outbreaks were reported in 2020), highlighting the need of new assessment methods. Herein we applied machine learning using the random forests method to evaluate and identify key points regarding the occurrence of Salmonella sp. along the Spanish food chain during 2015-2020, using data provided by the Spanish Agency for Food Safety and Nutrition. We compared the role of the three categorical variables [product (20 categories), region (18 categories) and stage (11 categories)]. Salmonella presence was influenced by the three explanatory variables considered: first by product, followed by region and stage. The most determinant product for Salmonella probability was 'meat', while the most important stage was 'slaughterhouse'. Specifically, the highest values were found in pig and poultry meats. In these products, the Salmonella probability was high at the early and final stages of the food chain, although not at intermediate stages. The presence of Salmonella in the final stages (retail) of the food chain is of concern, as it can cause human cases of salmonellosis, including outbreaks. This study demonstrates the utility of the random forest method to identify key points and evaluate the control efforts. We recommend improving the surveillance and control measures, especially in the product and stages pointed out by our analysis, and enhancing the data collection harmonization among the different autonomous communities.

Phylogenetic analyses of Salmonella detected along the broiler production chain in Trinidad and Tobago.

This study was conducted to determine the phylogenies of Salmonella strains isolated from cross-sectional studies conducted at hatcheries, broiler farms, processing plants, and retail outlets (broiler production chain) in Trinidad and Tobago over 4 yr (2016-2019). Whole-genome sequencing (WGS) was used to characterize Salmonella isolates. Core genome phylogenies of 8 serovars of public health significance were analyzed for similarities in origin and relatedness. In addition, Salmonella strains isolated from human salmonellosis cases in Trinidad were analyzed for their relatedness to the isolates detected along the broiler production chain. The common source of these isolates of diverse serovars within farms, within processing plants, between processing plants and retail outlets, and among farm-processing plant-retail outlet continuum was well-supported (bootstrap value >70%) by the core genome phylogenies for the respective serovars. Also, genome analyses revealed clustering of Salmonella serovars of regional (intra-Caribbean) and international (extra-Caribbean) origin. Similarly, strains of S. Enteritidis and S. Infantis isolated from human clinical salmonellosis in 2019 from Trinidad and Tobago clustered with our processing plant isolates recovered in 2018. This study is the first phylogenetic analysis of Salmonella isolates using WGS from the broiler industry in the Caribbean region. The use of WGS confirmed the genetic relatedness and transmission of Salmonella serovars contaminating chickens in broiler processing, and retailing in the country, with zoonotic and food safety implications for humans.

Isolation and identification of the broad-spectrum high-efficiency phage vB_SalP_LDW16 and its therapeutic application in chickens.

BACKGROUND: Salmonella infection in livestock and poultry causes salmonellosis, and is mainly treated using antibiotics. However, the misuse use of antibiotics often triggers the emergence of multi-drug-resistant Salmonella strains. Currently, Salmonella phages is safe and effective against Salmonella, serving as the best drug of choice. This study involved 16 Salmonella bacteriophages separated and purified from the sewage and the feces of the broiler farm. A phage, vB_SalP_LDW16, was selected based on the phage host range test. The phage vB_SalP_LDW16 was characterized by the double-layer plate method and transmission electron microscopy. Furthermore, the clinical therapeutic effect of phage vB_SalP_LDW16 was verified by using the pathogenic Salmonella Enteritidis in the SPF chicken model. RESULTS: The phage vB_SalP_LDW16 with a wide host range was identified to the family Siphoviridae and the order Caudoviridae, possess a double-stranded DNA and can lyse 88% (22/25) of Salmonella strains stored in the laboratory. Analysis of the biological characteristics, in addition, revealed the optimal multiplicity of infection (MOI) of vB_SalP_LDW16 to be 0.01 and the phage titer to be up to 3 × 1014 PFU/mL. Meanwhile, the phage vB_SalP_LDW16 was found to have some temperature tolerance, while the titer decreases rapidly above 60 ℃, and a wide pH (i.e., 5-12) range as well as relative stability in pH tolerance. The latent period of phage was 10 min, the burst period was 60 min, and the burst size was 110 PFU/cell. Furthermore, gastric juice was also found to highly influence the activity of the phage. The clinical treatment experiments showed that phage vB_SalP_LDW16 was able to significantly reduce the bacterial load in the blood through phage treatment, thereby improving the pathological changes in the intestinal, liver, and heart damage, and promoting the growth and development of the chicken. CONCLUSIONS: The phage vB_SalP_LDW16 is a highly lytic phage with a wide host range, which can be potentially used for preventing and treating chicken salmonellosis, as an alternative or complementary antibiotic treatment in livestock farming.

Multistate reptile- and amphibian-associated salmonellosis outbreaks in humans, United States, 2009-2018.

Non-typhoidal Salmonella cause an estimated 1.4 million human illnesses, 26,000 hospitalizations and 400 deaths annually in the United States. Approximately 11% of these infections are attributed to animal contact. Reptiles and amphibians are known sources of salmonellosis; young children (aged <5 years) are disproportionately affected by reptile- and amphibian-associated salmonellosis (RAAS) outbreaks. We describe multistate RAAS outbreaks to characterize illnesses and inform prevention efforts. RAAS outbreaks were defined as ≥2 culture-confirmed human Salmonella infections with similar pulsed-field gel electrophoresis patterns and epidemiologic, laboratory or traceback evidence linking them to a common reptile/amphibian exposure. Data sources included the Animal Contact Outbreak Surveillance System; CDC Outbreak Response and Prevention Branch's outbreak management database; PulseNet, the national molecular subtyping network for foodborne disease surveillance in the United States; and the National Antimicrobial Resistance Monitoring System. Twenty-six RAAS outbreaks were reported during 2009-2018, resulting in 1465 illnesses and 306 hospitalizations. The outbreaks were associated with turtles (19), lizards (5), snakes (1) and frogs (1). Sixteen (61.5%) outbreaks were linked to small turtles (<4 inches), resulting in 914 illnesses. Forty-nine percent of outbreak-associated patients were aged <5 years. Of 362 patients/caregivers interviewed, 111 (30.7%) were aware that reptiles/amphibians can carry Salmonella. Among 267 patient isolates with antimicrobial susceptibility information, 20 (7.5%) were non-susceptible to ≥1 antibiotic used to treat human salmonellosis. RAAS outbreaks result in considerable morbidity, particularly among young children. Illnesses linked to small turtles are preventable through education, targeted outreach to caregivers and paediatricians, and when appropriate, enforcement. Historically, individual states and jurisdictions have enforced existing or promulgated new authorities to address outbreaks. Preventing future RAAS outbreaks requires addressing challenges related to the illegal sale/distribution of small turtles; and for legal reptile sales, providing information on RAAS risk to consumers at point of sale to support informed pet ownership decisions.

Inactivated pentavalent vaccine against mycoplasmosis and salmonellosis for chickens.

Mycoplasma and Salmonella are serious pathogens threaten the poultry industry. This study aimed to prepare and evaluate an inactivated pentavalent vaccine targeting bacteria, including Salmonella enterica serovar Typhimurium (ST), Salmonella enterica serovar Enteritidis (SE), Salmonella enterica serovar Kentucky (SK), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS), from locally isolated strains. The prepared vaccine was adjuvanted with Montanide ISA70 oil and then tested for safety, sterility, and potency. The vaccine efficacy was evaluated in 110 specific pathogen-free, 1-day-old chicks, which were divided into three groups as follows: 1) vaccinated group (50 birds), which was subdivided into five subgroups of ten birds each; 2) control positive (challenged) group (50 birds), which was subdivided into five subgroups of ten birds each; and 3) control negative (blank) group, which included ten birds. Chicks in group 1 were administered the first dose of vaccine at 7 d of age followed by a booster dose after 3 wk. At 3 wk after booster vaccination, the chicks who were administered the booster dose were challenged and kept under observation until the end of the experiment when the chicks were approximately 10 wk. Details of clinical symptoms, daily mortality, weights, and postmortem lesions; serum samples; cloacal swabs; and nasal swabs were collected during the experiment. The humoral immune response to the prepared pentavalent vaccine was assessed using enzyme-linked immunosorbent assay. Our findings revealed that the prepared vaccine showed high protective antibody titers against Salmonella and Mycoplasma with 100% efficacy and no mortalities (100% survival rate) were recorded in vaccinated and challenged birds. The vaccine reduced both clinical signs and bacterial shedding post challenge in vaccinated birds in comparison with control positive group. The prepared vaccine did not affect the body weight gain of the vaccinated birds in comparison with control negative birds. The current study concluded that locally manufactured inactivated pentavalent vaccine offers protection to birds and could be employed as an effective tool along with biosecurity measures to overcome mycoplasmosis and salmonellosis in layer and breeder chicken farms in Egypt.

A multi-jurisdictional outbreak of Salmonella Typhimurium infections linked to backyard poultry-Australia, 2020.

Zoonotic salmonellosis can occur either through direct contact with an infected animal or through indirect contact, such as exposure to an infected animal's contaminated environment. Between May and August 2020, a multi-jurisdictional outbreak of Salmonella Typhimurium (STm) infection due to zoonotic transmission was investigated in Australia. In total, 38 outbreak cases of STm with a median age of 5 years were reported. Epidemiological investigation showed contact with live poultry to be a common risk factor with most cases recently purchasing one-week old chicks from produce/pet stores. Traceback investigation of cases identified 25 product/pet stores of which 18 were linked to a single poultry breeder farm. On farm environmental sampling identified the same STm genotype as identified in cases. Whole genome sequencing of both environmental and human outbreak isolates found them to be highly related by phylogenetic analysis. This investigation describes the first documented widespread zoonotic salmonellosis outbreak in Australia attributed to backyard poultry exposure and identified potential risk factors and prevention and control measures for future outbreaks. Prevention of future outbreaks will require an integrated One Health approach involving the poultry industry, produce/pet store owners, animal healthcare providers, public health and veterinary health agencies and the public.

The control of poultry salmonellosis using organic agents: an updated overview.

Salmonellosis is a severe problem that threatens the poultry sector worldwide right now. Salmonella gallinarium and Salmonella pullorum (Fowl typhoid) are the most pathogenic serovars in avian species leading to systemic infection resulting in severe economic losses in the poultry industry. Nontyphoidal serotypes of Salmonella (Paratyphoid disease) constitute a public health hazard for their involvement in food poisoning problems in addition to their zoonotic importance. Also, Salmonella species distribution is particularly extensive. They resisted environmental conditions that made it difficult to control their spread for a long time. Therefore, the current review aimed to through light on Salmonellosis in poultry with particular references to its pathogenesis, economic importance, immune response to Salmonella, Salmonella antibiotics resistance, possible methods for prevention and control of such problems using promising antibiotics alternatives including probiotics, prebiotics, symbiotics, organic acids, essential oils, cinnamaldehyde, chitosan, nanoparticles, and vaccines.

Risk factors for hospitalization among adults aged ≥65 years with non-typhoidal Salmonella infection linked to backyard poultry contact.

OBJECTIVE: We describe the epidemiology of live poultry-associated salmonellosis (LPAS) and investigate potential risk factors associated with hospitalization among adults aged ≥65 years in the United States during 2008-2017. LPAS is a public health concern in the United States, especially among people with increased risk for hospitalization, such as older adults. SAMPLE: We analysed data from people aged ≥65 years with non-typhoidal salmonellosis who reported live poultry contact within seven days prior to illness onset. PROCEDURE: We used logistic regression to estimate the odds of hospitalization associated with several risk factors including types of live poultry contact exposures. RESULTS: LPAS among older adults in this analysis resulted in high hospitalization rates. Salmonella Hadar infection was associated with increased hospitalization. Among older adults with LPAS, 109 individuals of 127 (86%) reported contact with live poultry at their or someone else's residence, and 85 of 105 with available information (81%) reported owning poultry. CONCLUSIONS AND CLINICAL RELEVANCE: Additional infection prevention information and education targeted at poultry-owning older adults are needed to prevent illness and hospitalization.

Lactobacillus plantarum Restores Intestinal Permeability Disrupted by Salmonella Infection in Newly-hatched Chicks.

Salmonella infections in newly hatched chicks result in enteric and systemic diseases with a high mortality. Probiotics can improve the health of a host. The purpose of the present study was to investigate the effect of Lactobacillus plantarum LTC-113 on the gut permeability in the presence or absence of Salmonella (Salmonella Typhimurium) infection. Newly hatched chicks were randomly allocated to 4 treatments (i) NC (negative control); (ii) LAC (the L. plantarum LTC-113-treated group); (iii) SAL (the Salmonella-infected group), and (iv) LAC + SAL (the L. plantarum LTC-113-treated and Salmonella-infected group). Compared with the NC group, the intestinal permeability and claudin-2 (CLDN-2) were significantly increased, while mRNA levels of zonula occludens-1 (ZO-1) and claudin-5 (CLDN-5) were significantly decreased in the SAL group. However, these changes were eliminated in the LAC + SAL group. Additionally, numbers of Salmonella in liver, spleen and ceca were significantly reduced in the LAC + SAL group compared with the SAL group. Moreover, L. plantarum LTC-113 prevented the increase of inflammatory meditators myeloperoxidase (MPO), LITAF, IL-1ß, IL-6 and inflammation scores induced by Salmonella. These findings indicate that L. plantarum LTC-113 can protect hosts from Salmonella induced intestinal barrier disruption by regulating expression of tight junction genes and inflammatory meditators and decreasing Salmonella colonization.

Highly suspected cases of salmonellosis in two cats fed with a commercial raw meat-based diet: health risks to animals and zoonotic implications.

BACKGROUND: Feeding raw meat-based diets (RMBD) to companion animals raises public health concerns for both animals and humans. While considerable attention has been paid to bacterial contamination of commercial pet food, few literature studies have investigated foodborne disease in companion animals. Salmonellosis is reported to be infrequent in cats but no known data or studies estimating feline salmonellosis are available or large-scale epidemiological studies assessing Salmonella risk factors. CASE PRESENTATION: Two highly suspected cases of salmonellosis in two cats fed with a commercial frozen poultry RMBD are presented, for the first time from the same household. The clinical presentation, diagnostics, treatment and follow-up are reported and the zoonotic implications are discussed. CONCLUSIONS: This case highlights the health risks posed to both animals and owners by feeding RMBD to pets, and suggests that these risks should be considered by veterinary practitioners.

Bacteriophage cocktail for biocontrol of Salmonella in dried pet food.

Human salmonellosis has been associated with contaminated pet foods and treats. Therefore, there is interest in identifying novel approaches for reducing the risk of Salmonella contamination within pet food manufacturing environments. The use of lytic bacteriophages shows promise as a safe and effective way to mitigate Salmonella contamination in various food products. Bacteriophages are safe, natural, highly targeted antibacterial agents that specifically kill bacteria and can be targeted to kill food pathogens without affecting other microbiota. In this study, we show that a cocktail containing six bacteriophages had a broadspectrum activity in vitro against a library of 930 Salmonella enterica strains representing 44 known serovars. The cocktail was effective against 95% of the strains in this tested library. In liquid culture dose-ranging experiments, bacteriophage cocktail concentrations of ≥10(8) PFU/ml inactivated more than 90% of the Salmonella population (10(1) to 10(3) CFU/ml). Dried pet food inoculated with a mixture containing equal proportions of Salmonella serovars Enteritidis (ATCC 4931), Montevideo (ATCC 8387), Senftenberg (ATCC 8400), and Typhimurium (ATCC 13311) and then surface treated with the six-bacteriophage cocktail (≥2.5 ± 1.5 × 10(6) PFU/g) achieved a greater than 1-log (P < 0.001) reduction compared with the phosphate-buffered saline-treated control in measured viable Salmonella within 60 min. Moreover, this bacteriophage cocktail reduced natural contamination in samples taken from an undistributed lot of commercial dried dog food that tested positive for Salmonella. Our results indicate that bacteriophage biocontrol of S. enterica in dried pet food is technically feasible.

Contaminated commercial dehydrated food as source of multiple Salmonella serotypes outbreak in a municipal kennel in Tuscany.

The authors describe a large outbreak of canine salmonellosis in a municipal kennel in Tuscany. During the outbreak, 174 samples of 'diarrhetic' and 'normal' faeces and two batches of commercial dehydrated dog food were cultured for pathogenic bacteria. The results of 25, out of a total of 41 dogs (60.9%) revealed at least one faecal sample as being positive for Salmonella; incidence per sampling ranged from 12.5% to 34%. Nine of 10 samples of dehydrated food were positive. Ten totally different serotypes were isolated from dry food and faeces: the results of the pulsed-field gel electrophoresis referred to similarity between the Salmonella Montevideo, Muenster and Worthington isolates recovered from both the food and canine faecal samples.

The prevalence of Salmonella enterica in Spanish feed mills and potential feed-related risk factors for contamination.

A cross-sectional study was conducted in Spain to estimate the prevalence of Salmonella enterica in feed mills and to identify and evaluate potential risk factors associated with feed contamination. A total of 3844 samples were collected from 523 different feed mills using a stratified sampling method. Samples were tested for the presence of Salmonella using conventional culture methods. When the presence of Salmonella was detected, samples were further characterised using serotyping at the National Reference Laboratory (NRL) for animal feed. Additional data about the biosecurity and hygiene measures, feed material used and compound feed produced, were collected by official veterinarians using a questionnaire in situ. In 144 of the feed mills visited (28%), Salmonella were present. However, it was only isolated from 4.8% of samples taken from all of the feed mills (3.5% from feed materials, 3.2% from compound feed and 12.5% from dust of the feed mill facilities). Salmonella serovars of public health importance (Enteritidis, Typhimurium, Infantis, Virchow and Hadar), were detected in only 2.7% of feed mills and in 0.3% of the samples studied. Logistic regression was used to investigate potential feed-mill risk factors for the isolation of Salmonella. Feed mill intake pits were demonstrated to have an increased risk of culture-positive dust samples (OR=6.4; 95% CI: 2.7-15.1). The feed material used in the production of compound feed was associated with recovery of Salmonella. Of the feed material used, cotton seeds were identified as having the highest odds of contamination (OR=3.8; 95% CI: 1.7-8.3). Pelleting appears to reduce the chance of contamination because non-pelleted compound feed is 8 times more likely to be contaminated than pelleted compound feed (OR=8.2; 95% CI: 2.5-26.6). The role of the feed itself in the epidemiology of Salmonella seems to be of limited importance as compound feed is not frequently contaminated at the feed mill level. This should not preclude Salmonella control measures from including all stages of feed production and they should have a risk-based approach according to the findings of this study.

Multistate outbreak of human Salmonella infections caused by contaminated dry dog food--United States, 2006-2007.

During January 1, 2006-December 31, 2007, CDC collaborated with public health officials in Pennsylvania, other states, and the Food and Drug Administration (FDA) to investigate a prolonged multistate outbreak of Salmonella enterica serotype Schwarzengrund infections in humans. A total of 70 cases of S. Schwarzengrund infection with the outbreak strain (XbaI pulsed-field gel electrophoresis [PFGE] pattern JM6X01.0015) were identified in 19 states, mostly in the northeastern United States. This report describes the outbreak investigation, which identified the source of infection as dry dog food produced at a manufacturing plant in Pennsylvania. This investigation is the first to identify contaminated dry dog food as a source of human Salmonella infections. After handling pet foods, pet owners should wash their hands immediately, and infants should be kept away from pet feeding areas.

Feed-borne outbreak of Salmonella cubana in Swedish pig farms: risk factors and factors affecting the restriction period in infected farms.

In 2003, a feed-borne outbreak of Salmonella Cubana occurred in Sweden as a result of contamination in a feed plant. Salmonella Cubana was detected in 49 out of 77 pig farms having received possibly contaminated feed. In this study, potential risk factors for farms being salmonella positive were examined, and a survival analysis was performed to investigate risk factors affecting the restriction period for salmonella positive farms. The median restriction time for all 49 farms was 17 weeks. An increased risk for farms being salmonella infected (positive in feed and/or faeces) was seen for fattening farms and farms feeding soy. The survival analysis showed that herds with a low level of infection and farms with a high hygiene level had shorter restriction times. This study is unique as it investigates a real outbreak of feed-borne salmonella, where the source of infection was reliably identified, the period of exposure could be defined and data were collected from all exposed farms.

Outbreak of Salmonella Dublin-associated abortion in Danish fur farms.

Outbreaks of Salmonella Dublin infections were recorded in 25 Danish mink and fox farms. All farms suffered extensive disease problems; clinical and pathological observations included abortion, stillbirths, necrotizing endometritis, and increased mortality. By genotyping with pulsed-field gel electrophoresis and amplified fragment length polymorphism, all isolates of S. Dublin had indistinguishable patterns. The outbreaks took place during April and May, around the time of whelping. During this period, mink are particularly susceptible to Salmonella infections. All affected farms were served by the same feed factory and it was concluded that a batch of contaminated feed was responsible for the outbreaks, although repeated culture of feed samples collected during the same period were negative. No other likely source could be identified. The results emphasize the importance of strict hygiene measures at feed factories and the proper use of ingredients of known Salmonella status, in particular during the whelping season. Infected mink farms did not have a higher risk of outbreak of salmonellosis in the year following the outbreak.